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HISTOLOGY – INTRODUCTION
HISTOLOGY
▲ Study of tissues of the body and how these tissues are arranged to constitute organs ▲ Focuses on HOW CELL’S STRUCTURE & ARRANGEMENT optimizes functions specific to each organ ▲ Scientific study of MICROSCOPIC STRUCTURES of tissues and organs of the body.
2 INTERACTING COMPONENTS OF TISSUES:
- CELL – produce ECM
- ECM (ExtraCellular Matrix) ▲ Consist of many MACROMOLECULES which form complex structures like COLLAGEN FIBRILS. ▲ Supports the cells ▲ Contains FLUID transporting nutrients into the cell and carrying away their waste and secretory products
TISSUE PREPARATION
PREPARATION OF TISSUE SLICES/SECTIONS ▲ Most common procedure used in histologic research ▲ So that it can be examined visually with transmitted light
PRESERVED ▲ IDEAL MICROSCOPIC PREPARATION so that the tissue on the slide will have the SAME STRUCTURAL FEATURES as it had on the body. ▲ NOT FEASIBLE because tissue preparation also REMOVES CELLULAR LIPID & cause slight distortion
BASIC STEPS FOR TISSUE PREPARATION FOR LIGHT MICROSCOPY
- FIXATION ▲ (^) 1st step in tissue preparation ▲ Small pieces of tissues are placed on a solution of chemicals that CROSS LINK PROTEINS and INACTIVATE DEGRADATIVE ENZYMES which PRESERVE CELL & TISSUE STRUCTURES. ▲ PURPOSE: TO PRESERVE ▲ Used to: Terminate cell matabolism Prevent enzymatic degradation of cells and tissues by AUTOLYSIS Kills pathogenic microorganisms Harden the tissue as a result of either CROSS LINKING or DENATURING of PROTEIN MOLECULES ▲ FIXATIVE - Solution of STABILIZING or CROSS LINKING COMPOUNDS ▲ FORMALIN - Commonly used fixative - 37% aqueous solution of FORMALDEHYDE - Preserves structures by REACTING with AMINO GROUP of PROTEINS (Cross linked LYSINE residues)- SAME WITH GLUTARALDEHYDE - Formalin DOES NOT ALTER the 3D structure of proteins so it maintains its ability to REACT with SPECIFIC ANTIBODIES ■ Important in IMMUNOCYTOCHEMICAL STAINING methods - POOR FIXATIVE OF CELL MEMBRANES – because it DOES NOT REACT WITH LIPIDS!
▲ GLUTARALDEHYDE
- Fixative for TEM ▲ OSMIUM TETROXIDE
- Used after glutaraldehyde
- Preserves and stains CELLULAR LIPIDS as well s PROTEINS
- Glutaraldehyde-treated tissues are immersed on this to preserve additional “ULTRASTRUCTURAL” detail ▲ FROZEN SECTIONS OF FORMALIN-FIXED TISSUE & DYES - To retain NUETRAL THAT DISSOLVE IN FATS LIPIDS ▲ FIXATIVES CONTAINING HEAVE METALS that bind to the PHOSPHOLIPIDS such as PERMANGANATE and OSMIUM TETROXIDE
- WASHING (based on PAWLINA)
- DEHYDRATION ▲ Tissue is transferred to a series of INCREASING CONCENTRATED ALCOHOL SOLUTION (ETHANOL), endinf in 100% which removes ALL THE WATER ▲ PURPOSE: TO REMOVE WATER
- CLEARING ▲ Alcohol is removed in organic solvent in which both alcohol and paraffin are miscible ▲ XYLOL or TOLUOL – most commonly used organic solvents
- INFILTRATION ▲ Tissue is placed in a MELTED PARAFFIN until it becomes completely infiltrated with the substance. ▲ Placed in an OVEN of 52- 60 C which evaporates the clearing solution
- EMBEDDING ▲ The paraffin-infiltrated tissue is placed in a small MOLD OF MELTED PARAFFIN and allowed to HARDEN ( for light microscopy) ▲ EPOXY RESINS/ PLASTIC RESINS
- Used for embedding in TEM
- Higher temperatures are AVOIDED unlike in paraffin which helps AVOID TISSUE DISTORTION ▲ PARAFFIN BLOCK – resulting product
- TRIMMING ▲ The resulting paraffin block is TRIMMED TO EXPOSE THE TISSUE FOR SECTIONING/SLICING ON A MICROTOME ( for Light microscopy)
NOTE: The resulting sections are MOUNTED ON A GLASS SLIDE USING MOUNTING MEDIUM ▲ Mounting Mediums -> PINENE/ ACRYLIC RESINS
COMPARISON BETWEEN LIGHT MICROSCOPY & TRANSMISSION ELECTRON MICROSCOPY (TEM)
LIGHT MICROSCOPY TEM FIXATIVE: FORMALIN GLUTARALDEHYDE + OSMIUM TETROXIDE INFILTRATION: PARAFFFIN EPOXY/PLASTIC RESINS
SLICING: MICROTOME
- 3-10 um thick
- STEEL edge knife
ULTRAMICROTOME
- < 1um thick
- GLASS/DIAMOND
Edge knife
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▲ Entire process of preparation & evaluation may take as little as 10 MINUTES ▲ RESULT TIME DEPENDS ON:
- Transport time of tissue from OR to pathology lab
- Pathologic technique used
- Experience of pathologist
STAINING
▲ Done cause cells & extracellular materials are COLORLESS ▲ Done to make tissues distinguishable from one another
CHEMICAL BASIS OF STAINING
H&E – most commonly used dye/stain in histology
▲ Dyes can be ACIDIC or BASIC compounds
BASIC DYES ACID DYES
-carries POSITIVE charge
-Binds to ANANION (- charge) - BASOPHILIC
-carries NEGATIVE charge
- Attracts CATION ANIONIC COMPONENTS: BASOPHILICS
PHOSPHATE GROUPS OF NUCLEIC ACIDS
-Heterochromatin -Nuclei of the Nucleus
-Cytoplasmic components such as ERGASTOPLASM (ionized phosphate groups in RNA SULFATE GROUPS OF GLYCOSAMINOGLYCANS -Extracellular materials (Complex carbohydrates of The matrix of CARTILAGE CARBOXYL GROUPS OF PROTEINS
CATIONIC COMPONENTS: ACIDOPHILIC
AMINO GROUP OF PROTEIN
-Extracellular Fibers CYTOPLASMIC FILAMENTS esp of
MUSCLE CELLS INTRACELLULAR MEMBRANOUS COMPONENTS & UNSPECIALIZED CYTOPLASM
BASIC DYES
TOLUIDIN BLUE
ALCIAN BLUE
METHYLENE BLUE
METHYLENE GREEN
PYRONIN G
HEMATOXYLYN – used with a MORDANT (intermediate link bet TISSUE & DYE)
ACIDIC DYES
EOSIN
ORANGE G
ACID FUSCHSIN
ANILINE BLUE
REACTION OF THE ANIONIC GROUPS
VARIES WITH PH
HIGH PH (about 10) – all 3 groups are ionized & a Available for reaction by
MALLORY STAINING TECHNIQUE
ANILINE BLUE – stain COLLAGEN ACID FUCHSIN – ORDINARY
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Eletrostatic linkages
SLIGHTLY ACIDIC TO NEUTRAL PH (5-7) -only SULFATE & PHOSPHATE LOW PH (BELOW 4) – only SULFATE GROUPS
CYTOPLASM, NUCLEI
ORANGE G - RBC
▲ HEMATOXYLIN & EOSIN
HEMATOXYLIN EOSIN
STAINS:
DNA in the NUCLEUS RNA-rich portion of Cytoplasm
Matrix of the cartilage
STAINS:
Other cytoplasmic structures & collagen
DARK BLUE or PURPLE PINK
COUNTERSTAIN
COUNTERSTAIN ▲ Single dye applied separately to distinguish additional features of a tissue
TRICHROME ▲ Allow greater distinctions among various extracellular tissue components ▲ More complex procedure
METACHROMASIA ▲ Absorbance change wherein certain BASIC DYES react with tissue components that SHIFT their normal color from BLUE to RED to PURPLE. ▲ Because of the presence of POLYANIONS within the tissue such as: Ground substance of CARTILAGE contains HIGH CONCENT Heparin-containing granules of MAST CELLS > of IONIZED PHOSPHATE & Rough Endoplasmic Reticulum of PLASMA CELLS SULFATE GROUPS
▲ So TOLUIDINE BLUE will appear PURPLE to RED when it stains, NOT BLUE!!!
ALDEHYDE GROUPS & SCHIFF REAGENTS
▲ ALDEHYDES contain CHO which is found in CARBOHYDRATES ▲ BASIS: Ability of SCHIFF REAGENT (Basic Fuchsin) to react with ALDEHYDE groups which results in RED color ▲ (^) Stains RED or MAGENTA
PERIODIC ACID-SCHIFF (PAS) REACTION
▲ Stains CARBOHYDRATES and CARBOHYDRATE-RICH MACROMOLECULES ▲ Used to demonstrate:
- Glycogen in cells
- Mucuos in various cells & tissues
▲ All are based on the interaction of light with tissue components and are used to reveal and study tissue features.
- BRIGHT-FIELD MICROSCOPY ▲ Used by most students and researchers ▲ Direct descendant of the microscopes that became widely available in 1800s ▲ Opened the 1 st^ major era of histologic research ▲ Stained tissue is examined through ORDINARY LIGHT passing through the preparation.
Bright-field microscope consists of:
a. LIGHT SOURCE ▲ For illumination of the specimen ( ex. Substage lamp)
b. (^) CONDENSER LENS ▲ To FOCUS the beam of light at the level of the specimen. ▲ FOCUSES LIGHT on the object to be studied
c. STAGE ▲ On which the slide or other specimen is placed
d. OBJECTIVE LENS ▲ To GATHER the light that has passed through the specimen ▲ ENLARGING and PROJECTING ▲ Example of Objective Lens:
X4 – for large area or field of tissue at LOW magnification X10 – for MEDIUM Magnification of smaller field X40 – for HIGH magnification of more detailed areas
e. OCULAR LENS ▲ Through which the image formed by the objective lens may be examined directly. ▲ Further magnification of image for another X10 (X40, X100, X400)
TOTAL MAGNIFICATION ▲ Obtained by MULTIPLYING the magnifying power of the OBJECTIVE LENS and OCULAR LENS. ▲ TM = OL X OL
NOTE:
ORGANS are THREE-DIMENSIONAL, HISTOLOGIC SECTIONS are ONLY TWO- DIMENSIONAL!
ARTIFACT ▲ Error in the preparation process ▲ Minor structural abnormalities ▲ Examples:
- Minor SHRINKAGE of cells or tissue regions produced by fixative, ethanol, heat from paraffin embedding
- Small WRINKLES in the section (Confused with linear structure in tissue)
- PRECIPITATES from the stain ( confused with cellular structures such as cytoplasmic granules)
- PHASE CONTRAST MICROSCOPY ▲ Enables examination of UNSTAINED CELLS and TISSUES ▲ Especially useful for LIVING CELLS and TISSUES ▲ (^) Used to enhance the contrast WITHOUT STAINING ▲ Used with LIVING CLUTURED CELLS ▲ Uses lens system that produces visible images from transparent objects (bcoz unstained cells and tissues are TRANSPARENT & COLORLESS) ▲ Based on the principle that light changes its SPEED when passing through cellular and extracellular structures with different REFRACTIVE INDICES.
▲ Takes advantage of small differences in REFRACTIVE INDEX in different parts of a cell or tissue sample. ▲ These changes cause the structures to appear LIGHTER OR DARKER. ▲ DARK PORTIONS = DENSE PORTION OF THE SPECIMEN ▲ (^) PROMINENT TOOL in ALL CELL CULTURE LAB bcoz it allows examination WITHOUT FIXATION or STAINING.
2 MODIFICATIONS OF PHASE CONTRAST:
1. INTERFERENCE MICROSCOPE
▲ Allows QUANTIFICATION of TISSUE MASS
- DIFFERENTIAL INTERFERENCE MICROSCOPE (DIM) ▲ Uses NOMARSKI OPTICS ▲ Useful for ASSESSING SURFACE PROPERTIES of cells and other biologic objects. ▲ Produces an image of LIVING CELLS with a more apparent 3D ASPECT
- (^) DARK-FIELD MICROSCOPY ▲ NO DIRECT LIGHT from the light source is gathered by the OBJECTIVE LENS ▲ ONLY light that has been scattered or diffracted by structures in the specimen reaches the objective. ▲ Useful in examining autoradiographs in which developed SILVER GRAINS appear WHITE in DARK BACKGROUND ▲ Useful in examining URINE for CRYSTALS (such as URIC ACID & OXALATE) and demonstrating specific bacteria such as SPIROCHETES particularly TREPONEMA PALLIDUM, microorganism that causes SYPHILIS, a STD.
- FLUORESCENCE MICROSCOPY ▲ Make use of the ability of certain molecules to fluoresce under ultraviolet light. ▲ A molecule that fluoresce emits light of wavelengths in the visible range when exposed to an Ultraviolet (UV) source. ▲ Tissue sections are irradiated by UV light and emission is in the visible portion of the spectrum. ▲ Fluorescent substances appear BRIGHT in dark environment/ background. ▲ Used to display naturally occurring fluorescent molecules such as VITAMIN A and NEUROTRANSMITTERS ▲ Used in introduced fluorescence for detection of ANTIGEN or ANTIBODIES in IMMUNOCYTOCHEMICAL staining method
FLUORESCENT COMPOUNDS/DYES:
a. FLUORESCEIN ▲ Most commonly used dye and absorbs UV light & emits GREEN light b. ACRIDINE ORANGE ▲ Binds both DNA & RNA ▲ DNA -> YELLOW light ▲ RNA -> ORANGE light c. DAPI & HOECHST stains ▲ Specifically bind DNA & used to stain CELL NUCLEI ▲ Emits BLUE light under UV d. FLUORESCEIN-PHALLOIDIN ▲ Binds with ACTIN FILAMENTS & stain them GREEN.
- ULTRAVIOLET MICROSCOPE ▲ Uses quartz lenses with an ultraviolet light source ▲ Image depends on the absorption of UV light by molecules in the specimen. ▲ UV SOURCE wavelength -> 200 nm ▲ UV microscope resolution -> 0.1 um ▲ Specimen cannot be inspected directly through an ocular lens because UV light is NOT VISIBLE and is INJURIOUS to the eye.
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A BEAM OF ELECTRONS focused using ELECTROMAGNETIC LENSES “PASSES THROUGH” the tissue sections to produce an image with BLACK, WHITE & intermediate shades of GRAY which corresponds to:
a. Areas that are BRIGHTER/ ELECTRON LUSCENT
- Electrons pass readily b. Areas that are DARKER/ ELECTRON DENSED
- Electrons are ABSORBED or DEFLECTED
▲ TISSUE PREPARATION FOR TEM:
- GLUTARALDEHYDE – preserves PROTEINS by cross linking them
- OSMIUM TETROXIDE – HEAVY METAL imparts electron density to cells; reacts with LIPIDS particularly PHOSPHOLIPIDS
- EPOXY RESIN – used for infiltration & embedding instead of Paraffin
- ULTRAMICROTOME – contains DIAMOND KNIVES used to section the specimen
▲ HEAVY METAL IONS that may be added to the tissues for TEM:
- OSMIUM TETROXIDE – binds to LIPIDS particularly PHOSPHOLIPIDS
- URANYL NITRATE – added to ALCOHOL sol’n in dehydration to increase the density of components of cell junctions and other sites
- LEAD CITRATE – same with Uranyl
▲ CRYOFRACTURE & FREEZE ETCHING
- Techniques that allow TEM to study cells WITHOUT FIXATION or EMBEDDING
- Same process with freezing in light microscopy
- Uses LIQUID NITROGEN
▲ FRACTURE PLANE
- Passes preferentially through the HYDROPHOBIC PORTIONS of the PLASMA MEMBRANE, exposing the interior of the plasma membrane.
- Resulting fracture produces 2 surfaces:
- E- FACE -> Surface back by EXTRACELLULAR SPACE
- P- FACE -> Backed by Protoplasm (Cytoplasm)
B. SCANNING ELECTRON MICROSCOPY ▲ Almost the same with TEM except that the Beam DOES NOT PASS THROUGH the specimen but is scanned across its surface ▲ Provides high resolution view of the surfaces of cell, tissues and organs ▲ Surface of specimen is dried and spray-coated with a layer of heavy metal (GOLD) which reflects electrons in beam scanning the specimen. ▲ Easier to interpret because it presents a 3D view.
SCANNING-TRANSMISSION ELECRON MICROSCOPY ▲ Combines features of the TEM & SEM to allow ELECTRON PROBE X-RAY MICROANALYSIS.
ATOMIC FORCE MICROSCOPY (AFM) ▲ One of the MOST POWERFUL TOOLS for studying the SURFACE TOPOGRAPHY at molecular and atomic resolution. ▲ NON-OPTICAL MICROSCOPE – works same way as fingertips but uses an ultrasharp, pointed PROBE (CANTILEVER) that is dragged across the surface of the specimen. ▲ CONTACT MODE and TAPPING MODE ▲ Resolving power = 50 PM ▲ MAJOR ADVANTAGE OF AFM: Specimen DOES NOT HAVE TO BE IN A VACUUM unlike TEM or SEM (can be in WATER) so it is feasible to image LIVING CELLS & their SURROUNDING ENVIRONMENTS.
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VIRTUAL MICROSCOPY
▲ Digital procedure that is alternative to the examination of glass slides using a light microscope. ▲ Permits study of tissues using a computer or other digitl devices WITHOUT an ACTUAL STAINED SLIDE or MICROSCOPE ▲ VIRTUAL SLIDE ▲ Digital representation of a glass slide which can be viewed remotely without a light microscope.
